bioRxiv preprint doi: https://doi.org/10.1101/610170; this version posted April 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Identification of new viruses specific to the honey bee mite
Varroa destructor
Salvador Herrero*, Sandra Coll, Rosa M. González-Martínez, Stefano Parenti,
Anabel Millán-Leiva, Joel González-Cabrera*
ERI BIOTECMED. Department of Genetics. Universitat de València, Valencia, Spain.
*Corresponding authors:
Email: [email protected], tel: +34 96 354 3006
Email: [email protected], tel: +34 96 354 3122
Keywords: iflavirus, picornavirus, insobevirus, qPCR, +ssRNA virus
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Abstract
Large-scale colony losses among managed Western honey bees have become a
serious threat to the beekeeping industry in the last decade. There are multiple
factors contributing to these losses but the impact of Varroa destructor parasitism is
by far the most important, along with the contribution of some pathogenic viruses
vectored by the mite. So far, more than 20 viruses have been identified infecting the
honey bee, most of them RNA viruses. They may be maintained either as covert
infections or causing severe symptomatic infections, compromising the viability of the
colony. In silico analysis of available transcriptomic data obtained from mites
collected in the USA and Europe as well as additional investigation with new
samples collected locally allowed the description of three novel RNA viruses. Our
results showed that these viruses were widespread among samples and that they
were present in the mites and in the bees but with differences in the relative
abundance and prevalence. However, we have obtained strong evidence showing
that these three viruses were able to replicate in the mite, but not in the bee,
suggesting that they are selectively infecting the mite. To our knowledge, this is the
first demonstration of Varroa-specific viruses, which open the door to future
applications that might help controlling the mite through biological control
approaches.
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Introduction
The ectoparasitic mite Varroa destructor Anderson and Trueman (Arachnida: Acari:
Varroidae) is considered a major driver for the seasonal losses of managed Western
honey bee (Apis mellifera L.) colonies reported worldwide (Rosenkranz et al. 2010).
There are multiple factors contributing to these losses, but the impact of V.
destructor is by far the most important in many cases (Roberts et al. 2017). The
damage caused by the mite is a result of the combined effect of direct feeding on the
fat body of immature and adult bees (Ramsey et al. 2019) and the transmission of an
extensive set of viruses that infect and debilitate them (McMenamin and Genersch
2015). This resulted in reduced immune response, low tolerance to pesticides,
impaired mobility or flying capacity, reduced weight, morphological deformities,
paralysis, etcetera (De Jong et al. 1982; Yang and Cox-Foster 2007; Yang and Cox-
Foster 2005).
So far, more than 20 viruses have been identified in the honey bees (McMenamin
and Genersch 2015), most of them ssRNA viruses. Among them, the Acute bee
paralysis virus (ABPV), Deformed wing virus (DWV), Israel acute paralysis virus
(IAPV), Kashmir bee virus (KBV), Sacbrood virus (SBV), Slow bee paralysis virus
(SBPV), Chronic bee paralysis virus (CBPV), Lake Sinai virus (LSV), Kakugo virus,
Varroa destructor virus-1 and Black queen cell virus (BQCV) (Boecking and
Genersch 2008; Di Prisco et al. 2011; McMenamin and Genersch 2015; Santillán-
Galicia et al. 2014) . However, in absence of V. destructor parasitism, many of these
viruses have been found infecting seemingly healthy honey bee colonies, in what are
considered as covert infections, that do not cause any detectable clinical impact on
them (McMenamin and Genersch 2015). After the host shift of V. destructor from
Apis cerana L. to A. mellifera L., the prevalence and pathogenicity of some of the
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aforementioned viruses have increased significantly (Rosenkranz et al. 2010). This
combination of viruses’ pathogenicity and the high vectoring capacity of V. destructor
is considered for many as one of the main causes of the Colony Collapse Disorder
(CCD), responsible for very high losses of colonies in Europe and the USA (Potts et
al. 2010; Vanengelsdorp et al. 2009).
Thus, it is well documented the ‘relation’ of V. destructor with bee pathogenic viruses
but there is very little information available about viruses specific (or pathogenic) for
the mite itself. There are no evidences of bee pathogenic viruses causing any impact
on V. destructor physiology, even with high loads of some of them recorded in mite
virome. There are even viruses discovered in the mite whilst showing no evidence of
pathogenicity for it (i.e. VdV-1) (Ongus et al. 2004). Recent research has identified
two novel viruses present in V. destructor but not detected in the bees. However, the
information available does not show clear evidences of the possible differential
specificity of these viruses for V. destructor or the honey bee (Levin et al. 2016).
In this paper, we report the identification and phylogenetic characterization of three
new viruses in the virome of V. destructor. We show strong evidence indicating that
these viruses can replicate in the mite but not in the bee, suggesting that they are
selectively infective for the mite without affecting the replication of DWV.
Material and Methods
Mite samples
Mites used in this study were collected directly from the brood of honey bee combs,
from hives located nearby Valencia, Spain. Capped cells were opened with a pair of
tweezers to extract immature bees and the mites parasitizing them. Bees and mites
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extracted from the same cell were put together in a microcentrifuge tube, snapped
frozen in liquid nitrogen and stored at -80 ºC until used for qPCR analysis.
Virus discovery and genome sequence analysis
Viral sequences were identified in two V. destructor transcriptomes assembled from
data downloaded from the SRA (https://www.ncbi.nlm.nih.gov/sra). The mites used
to generate these data were collected in the USA (Accession SRS353274) and in the
UK (Accession PRJNA531374). A set of invertebrate picornaviral sequences from
different families was used as query in Blastx search against the different
transcriptomes. Those sequences producing significant hits were manually curated
to remove redundant sequences. Sequences with similarity restricted to functional
domains like zinc finger domain, helicase domain, and RNA binding domain were
removed since they can be in viral as well as in non-viral proteins, and possibly
representing non-viral sequences.
The genomic structure, gene content, and location of the conserved motifs for the
non-structural proteins (helicase, protease and RNA-dependent RNA polymerase),
were obtained by comparison with their closest viral species.
Phylogenetic analysis
RNA-dependent RNA polymerase was selected to establish the phylogenetic
relationship of viruses described in this study with the closest viruses and
representative members of nearest viral families, retrieved from NCBI GenBank
(www.ncbi.nlm.nih.gov/genbank).
Protein sequences comprising putative conserved domains for +ssRNA viruses were
aligned using MAFFT version 7.409 software employing the E-INS-i algorithm
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(Standley and Katoh 2013). Alignments were examined by eye and subsequently,
ambiguously aligned regions were removed using TrimAl program (Capella-Gutiérrez
et al. 2009). The most appropriate evolutionary model (best-of-fit model) according
with corrected Akaike Information Criterion (AICc) was calculated using Small
Modelling Selection software (Longueville et al. 2017). Phylogenetic trees were
inferred by maximum likelihood approach implemented in PhyML version 3.1
(Guindon et al. 2010), with the LG substitution model, empirical amino acid
frequencies, and a four-category gamma distribution of rate, and using Subtree
Pruning and Regrafting (SPR) branch-swapping. Branch support was assessed
using the approximate likelihood ratio test (aLRT) with the Shimodaira–Hasegawa-
like procedure as implemented in PhyML. Branches with <0.6 aLRT support were
collapsed using TreeGraph software (Stöver and Müller 2010).
Virus detection and quantification
Presence and abundance of RNA viruses in honey bee and V. destructor samples
were determined by detection of viral RNA genomes using reverse transcription
quantitative real-time polymerase-chain reaction (RT-qPCR). For this purpose, total
RNA was isolated from individual V. destructor and honey bees using Trizol reagent
(Sigma Aldrich). For the mites, each individual was homogenized with a plastic
pestle in microcentrifuge tubes containing 100 µl of Trizol in two cycles of freeze and
thaw in liquid nitrogen. After that, 100 µl of Trizol were added to the mixture and the
RNA extraction continued following the manufacturer’s protocol. Total RNA from
honey bees was isolated from the abdomen of individual bees after homogenization
in 200 µl of Trizol reagent using the TissueLyser LT (Qiagen N. V.) for 3 min. For
cDNA synthesis, 300 ng of each RNA was reverse transcribed to cDNA using
random hexamers and oligo(dT) primers and following the instructions provided in
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the PrimeScript RT Reagent Kit (Perfect Real Time from Takara Bio Inc., Otsu
Shiga, Japan). RT-qPCR was carried out in a StepOnePlus Real-Time PCR System
(Applied Biosystems, Foster City, CA). All reactions were performed using 5x HOT
FIREpol EvaGreen qPCR Mix Plus (ROX) from Solis BioDyne (Tartu, Estonia) in a
total reaction volume of 20 μl.
Forward and reverse primers used in the study were either described elsewhere or
designed de novo for this study using the Prime3Plus software (Nijveen et al. 2007)
(Table 1). Primers efficiency were calculated for all primer pairs used in RT-qPCR
(Table 1). The ribosomal 18S gene (Campbell et al. 2016) and the ApiDorsal gene
(Di Prisco et al. 2016) were used as endogenous control of the RNA concentration
for the varroa and bee samples, respectively. As a preliminary outcome of the RT-
qPCR, the identity of amplified fragments was confirmed by Sanger sequencing. For
relative viral load quantification in each of the samples, the following equation was
used, Relative abundance = E (primers virus)-Ct (virus) / E (primers endogenous)-Ct
(endogenous), were E, refers to the efficiency of each pair of primers and Ct the values of
the Threshold Cycle obtained in the qPCRs. The limit of detection for each virus was
established based on the average Ct values obtained for the negative controls (total
RNA without cDNA synthesis). The Ct values equal or greater to the average Cts for
the controls were considered free of the studied virus and reported as non-detected
(ND).
Semi-quantitative RT-PCR was used to detect the negative RNA strands of the VdV-
5, VdV-3 and VdIV-2 in varroa and bees as previously described (Jakubowska et al.
2015; Llopis-Giménez et al. 2017). Briefly, tagged primer was used for the specific
synthesis of cDNA due to the occurrence of self-priming, which is often observed for
RNA viruses. Total RNA was extracted as described above from a pool of about 10
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varroa mites, or 10 bee heads or abdomens. For this, 500 ng of RNA were used for
cDNA synthesis using a tagged specific primer (Table S1). cDNA synthesis was
performed using the PrimeScript RT reagent kit from Takara Bio Inc (Otsu Shiga,
Japan) at 42 °C for 30 min, following the manufacturer’s protocol. Three microliters
of cDNA were used for subsequent PCR reactions using the tag region as a forward
primer, and specific reverse primers (Table S1). PCR was performed using the
following conditions: 95 °C for 5 min, followed by 35 cycles of annealing at 52 °C for
30 seconds, and elongation at 72 °C for 1 min using DreamTaq DNA polymerase
(Thermo Fischer Scientific, Waltham, MA, USA). Presence of viral positive strand
was confirmed in each of the samples used for the negative strand detection by RT-
qPCR as described above.
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Results and discussion
New viruses associated to V. destructor
The presence of virus-like sequences associated with V. destructor was investigated
via data mining using transcriptomic data available in public databases. These
analyses showed that among other viruses, typically associated with the vectoring
capacity of V. destructor (data not shown), the data contained sequences belonging
to three different +ssRNA viruses featuring conserved regions and motifs suggesting
that they were novel. Detailed analyses of the selected sequences revealed the
absence of mutations or indels generating premature stop codons, which supported
the idea that all three sequences belong to viruses actively infecting V. destructor
(Fig. 1-4). Two of the viruses showed significant similarity with a virus recently
described that was named Varroa destructor virus 3 (VdV-3) (Accession
KX578272.1) (Levin et al. 2016). Here, we named that virus VdV-3 ISR to
differentiate it from a closely related variant found in our data. This variant showed
90.8 % overall genome identity with VdV-3 ISR. Thus, we consider it a new variant of
the same species and we named it VdV-3 USA. The other virus similar to VdV-3 ISR
showed an overall identity of only 75.0 %. Therefore, we consider it a new species
and it was named Varroa destructor virus 5 (VdV-5) (Fig. 1A). Despite differences in
the amino acid sequence, the genome of the two viruses described here were very
similar in structure. Two coding Open Reading Frames (ORF) were predicted for
both viral genomes, ORF1 with 817 and 822 amino acids in VdV-3 USA and VdV-5,
respectively and ORF2 with 493 amino acids. In ORF1 we identified conserved
motifs generally found in closely related species: a transmembrane domain (TM), a
trypsin-like serine protease (Pro) and a viral genome-linked protein (VPg). On the
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other hand, ORF2 contained only conserved motifs for the RNA-dependent RNA
polymerase (RdRp) (Fig. 1A).
The phylogenetic analysis of the new viruses using the RdRp sequence showed that,
given the sequence similarity, they clustered together with VdV-3 ISR in a branch
containing the other unassigned invertebrate-derived viruses closely related to plant
+ssRNA viruses from the Solemoviridae (Sobemovirus, and Polemovirus) and
Luteoviridae (Enamovirus) families (Fig. 2). They are also closely related with
Barnaviruses, described in fungi. Members of this group have a similar genomic
structure with two ORFs spanning 3 to 4.2 kb. This group includes viruses and viral
sequences described in other acarine species like the deer tick (Ixodes scapularis)
and the American dog tick (Dermacentor variabilis) (Harvey et al. 2019; Levin et al.
2016; Levin et al. 2019; Pettersson et al. 2017; Sadeghi et al. 2018; Shi et al. 2016;
Webster et al. 2016) and not much information about their incidence and replication
in a specific host has been reported. Nevertheless, given the mentioned similarities
and the replicative activity of these viruses (see below), these V. destructor
associated viruses could define a new family of +ssRNA viruses infecting
invertebrates. Thus, we propose to name this new family as “Insobeviridae”.
The third viral sequence showed high similarity to the iflavirus Varroa destructor virus
2 described earlier (VdIV-2) (Levin et al. 2016), sharing 83.4 % of genome identity
between them (Fig. 1B, S1). Due to the high similarity with the VdV-2, we resolve
that they were genotypes/isolates from the same species, hence we named as
Varroa destructor iflavirus 2 UK (VdIV-2 UK), to discriminate it from the previous
described virus which we named as Varroa destructor iflavirus 2 ISR (VdIV-2 ISR).
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The genome of the VdIV-2 UK contains a single large ORF, that translates into 2996
amino acids polyprotein, displaying the characteristic Iflaviridae structure
organisation (Valles et al. 2017) (Fig 1B). Conserved motifs for the Non-structural
proteins, RdRp, 3C-like cysteine protease and RNA helicase, were detected in the
C-terminal region of the polyprotein. Capsid proteins were identified based on their
homology to those from other members of the family. Polyprotein sequence for the
two viruses showed a 91.3 % similarity, nonetheless, most amino acid variations
were concentrated on the Structural-proteins coding region, with half of the
differences (131 out of 262 total aa changes) on the tentative leader protein (L)
sequence, at the polyprotein N-terminus (Fig. 1B).
Viral incidence in field samples
To validate the actual presence of these viruses in the wild, we designed,
synthesized and tested primer pairs specific for each of the three viruses described
above (See Material and Methods). The detection was carried out in both, the V.
destructor individuals parasitizing immature bees (Fig. 3A) and also in these very
same bees collected from the same capped cell (Fig. 3B). The results showed that
the three viruses were detected in the mites as well as in the bees although with
differences in the relative abundance and prevalence. The virus VdV-5 was present
in all mites tested while it was only detected in 57 % of individual bees. Virus VdV-3
USA showed the lowest prevalence with detections in 36 % and 43 % of the
analysed mites and bees, respectively. On the other hand, the iflavirus, VdIV-2 UK,
was found in all tested mites and in most of the bees (86 %) (Fig. 3). It is interesting
to point out that in about 30 % of the samples it was possible to detect the three
viruses in a single individual (35 % in mites and 29 % in bees). The high incidence of
these viruses in our samples (from Spain) and the presence of closely related
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variants in samples collected in Israel (Levin et al. 2016), the UK and the USA
(transcriptomic data used in this study) is a clear indication of their widespread
distribution in the populations and that they are evolving, possibly to gain better
adaptation to different mite populations and environmental conditions. Whether these
new viruses influence the mite or bee performances or not, remain to be elucidated
in further studies. However, an important step to study its relevance in the varroa-
bee system is to determine their replication and host specificity.
Viral replication and specificity
A +ssRNA virus produces an intermediate negative-strand RNA when it replicates.
Thus, the detection of negative-strand viral RNA is indicative of viral replication and
supports its viral identity. We designed specific sets of primers to detect the negative
strand by PCR, to confirm the viral origin of the sequences as well as to determine
their host specificity. The results showed that although it was possible to detect viral
genomes in the bee heads and/or abdomens (Fig 4. Upper panels), the negative
strand for VdV-5, VdV-3, and VdIV-2 was only detected in the mites (Fig 4),
indicating that the sequences described here belong to active viruses and that they
replicate exclusively in the mites. It seems that their presence in the bees was
probably due to the typical exchange of fluids between the bee and the mite
parasitizing it.
In addition to the lack of viral negative strand in the bee samples, there are other
indirect evidences supporting the specific replication of these new viruses in mite
tissues. For example, it is possible to estimate the abundance of mite contents in the
parasitized bees from the amplification of the V. destructor endogenous gene Vd18S
in bee samples. We have observed a positive correlation (Pearson r = 0.44; P-value
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= 0.019) between the viral abundance in the bees and the amount of mite content
(Fig 5A). However, the comparison of viral loads in pairs bee/mite did not reveal a
correlation of relative abundance of each of the viruses between the mite and its
parasitized bee (P-value>0.05 for all the viruses) (Fig. 5B). In fact, it was remarkable
that, in some cases, the mite had a very high virus load while it was not possible to
detect the same virus in the bee (Fig. 5B). Accordingly, we hypothesised that the
presence of viral sequences in the bees is just circumstantial and does not reflect
viral load on the mite.
Previous evidences of the specific replication of these viruses in V. destructor were
based on the absence of RT-PCR amplification of viral sequences in the bees (Levin
et al. 2016). However, as they used adult worker bees from the colonies to make the
detection it is likely that these were not parasitized bees. Hence, there were not
exchange of fluids with the mites. In this study, as we used parasitized immature
bees, we detected the presence of the three viruses in a large proportion of the
analysed bees, but the absence of negative strain clearly indicates that the viruses
are not replicating in the bees. Absence of viral replication in the bees does not
directly imply the absence of direct or indirect effects on the parasitized bees. For
instance, viral particle in the bee tissues could boost or suppress the immunity of the
bees. Immunosuppression of honey bee has been associated with the presence of
bee-infecting viruses such as DWV (Di Prisco et al. 2016), however direct effects on
the bee of non-replicative viruses have not been reported. Alternatively, the
replication of these viruses could have an indirect effect interfering or promoting the
action of other viruses that replicate actively on the bees.
Interaction of the new viruses with DWV
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The synergistic interaction between V. destructor and DWV is one of the major
threats to the honey bee industry (Di Prisco et al. 2016). DWV belongs to the
Iflaviridae family (Valles et al. 2017) so it is also a +ssRNA virus, like the new viruses
described here. Therefore, it is possible that its replication and transmission of DWV
in the mites could be influenced by the presence of other viruses from the same
family (VdIV-2) or with similar replication mechanism (VdV-3 and VdV-5). To test for
this potential interference, we determined the abundance of DWV in the studied
varroa individuals and the possible correlation with the abundance of the new
viruses.
We have a positive detection of DWV in 100 % of the mites tested although with
differences in the relative abundance in each case (Fig. 6). To test if these
differences in the relative abundance of DWV were influenced by the new viruses,
the correlation in their relative abundance were analysed. Viral relative abundances
were compared by grouping together the insobeviruses (VDV-3 USA and VDV-5)
(Fig. 6A) on one hand and the iflavirus (VdIV-2 UK) on the other (Fig. 6B). No
correlation was found in any case (P-values >0.05), so it seems that the newly
detected viruses do not interfere with the presence and infection process of DWV
and vice versa.
Conclusion
Three new viruses infecting V. destructor have been detected in samples from
several locations. We have evidenced that these new viruses actively replicate in V.
destructor but not in A. mellifera, thus to our knowledge this is the first demonstration
of Varroa-specific viruses. Although further investigation would be needed to
determine the mechanisms of viral transmission and to measure whether they induce
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a deleterious effect on mite physiology, these new viruses may be used as part of
biological control approaches to contribute to the reduction of varroa parasitism on
the bee health.
Acknowledgments
Joel González-Cabrera was supported by the Spanish Ministry of Economy and
Competitiveness, Ramón y Cajal Program (RYC-2013-13834). The work at the
Universitat de València was funded by a grant from the Spanish Ministry of Economy
and Competitiveness (CGL2015‐65025‐R, MINECO/FEDER, UE). Research at the
Universitat de València of Stefano Parenti was possible thanks to the Erasmus+
program from the EU. Finally, we thank Fernando Calatayud and Enrique Simó, from
the local beekeeper association “apiADS”, for providing bees and mites.
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Figures legend
Figure 1. Schematic representation of the new viruses
Schematic representation of the genome organization for the Varroa destructor
viruses described on this project, compared to the closest previous described VdV-3
(1A) and VdIV-2 (1B) (accessions KX578272.1 and KX578271.1, respectively). Grey
boxes represent ORF and vertical black lines indicate amino acid variation.
Percentage values show amino acid sequence identities.
Predicted ORFs are shown as boxes. Conserved domains on the amino acid
sequence are represented by letters (RdRp: RNA-dependent RNA polymerase; Pro:
Protease; Hel: RNA Helicase; VP1 to VP4: Capsid proteins 1 to 4; L: Leader protein;
TM: Transmembrane domain; VPg: Viral genome-linked protein). Percentage of aa
identities are provided for the different domains of the polyprotein as well as for the
different ORFs.
Figure 2. Phylogenetic analysis of the new Insobeviruses and its relationship
with other RNA viruses
Maximum-likelihood phylogenetic tree of the putative RdRp amino acid sequences of
Varroa destructor virus 5 and 3, and closely related viruses. Values on blanches
indicates the aLRT support. Branches with aLRT values <